1. Field of the Invention
This invention relates to assay methods, and reagent means for use therein, of the homogeneous and heterogeneous specific binding type for determining qualitatively or quantitatively a ligand in, or the ligand binding capacity of, a liquid medium. In particular, the invention relates to an improved fluorescer-labeled specific binding assay.
In conventional specific binding assay techniques, a sample of the liquid medium to be assayed is combined with reagent means of various compositions. Such compositions include a labeled conjugate comprising a binding component incorporated with a label, which labeled conjugate participates with other constituents, if any, of the reagent means and the ligand or ligand binding capacity in the medium under assay to form a binding reaction system producing two species or forms of the labeled conjugate, a bound-species and a free-species. The relative amount or proportion of the labeled conjugate that results in the bound-species compared to the free-species is a function of the presence (or amount) of the ligand or ligand binding capacity to be detected in the test sample.
As an illustration, a conventional competitive binding assay technique for the detection of a specific ligand will now be described. In such a technique, the reagent means would comprise (1) a labeled conjugate in the form of the ligand to be detected (e.g., an antigen or hapten) chemically linked to a label, and (2) a specific binding partner for the ligand (e.g., an antibody). Upon combination of the test sample and the reagent means, the ligand to be detected and the ligand portion (i.e., the binding component) of the labeled conjugate would compete in a substantially nondiscriminating manner for noncovalent binding to the specific binding partner. As a result, either the amount of labeled conjugate that becomes bound to the binding partner (i.e., that which results in the bound-species) or that amount which remains free (i.e., unbound to the binding partner and thus that which results in the free-species) can be measured as a function of the amount of competing ligand present. The amount of labeled conjugate resulting in either species is determined by measuring, i.e., monitoring, the label therein.
Where the labeled conjugate in the bound-species is essentially indistinguishable in the presence of that in the free-species by the means used to monitor the label, the bound-species and the free-species must be physically separated in order to complete the assay. This type of assay is referred to in the art as "heterogeneous". Where the bound-species and free-species forms of the labeled conjugate can be distinguished in the presence of each other, a "homogeneous" format can be followed and the separation step avoided.
2. DESCRIPTION OF THE PRIOR ART
The first discovered type of highly sensitive specific binding assay was the radioimmunoassay which employs a radioactive isotope as the label. Such an assay necessarily must follow the heterogeneous format since the monitorable character of the label is qualitatively unchanged in the free- and bound-species. Because of the inconvenience and difficulty of handling radioactive materials, many new assay systems have been devised using materials other than radioisotopes as the label component, including enzymes, bacteriophages, metals and organometallic complexes, coenzymes, enzyme substrates, enzyme inhibitors, cyclic reactants, organic prosthetic groups, chemiluminescent reactants, and fluorescent molecules.
Those specific binding assays developed thus far which employ a fluorescent label rely upon photogenic excitation of the fluorescer, i.e., excitation of the fluorescer by means of irradiation with light to an energy state at which it emits light. The fluorescer can thus be measured by the resulting total or peak light emitted or some other detectable characteristic of the emitted light, such as its polarization. The requirement of incident light to initiate the measurement of the fluorescer is a quite disadvantageous feature of these prior art fluorescent binding assays. Measurement instrumentation is typically both mechanically and electronically complex in order to cope with the problem of interfering background light from incident radiation.
Other specific binding assays have evolved wherein the label is chemiluminescent, i.e., produces light upon chemical reaction. Incident radiation is not required to initiate the monitoring reaction in these assays, however, the sensitivity of such chemiluminescent binding assays presently known is theoretically limited because the material used as the label is invariably, in the prior art, a consumable reactant in the light producing reaction. Furthermore, the chemiluminescent-based specific binding assays thus far published are generally susceptible to protein quenching of light production.
The state of the art of the aformentioned fluorescent and chemiluminescent binding assays will now be briefly outlined with reference to specific prior art publications.
The basic concept of employing fluorescers as labels in specific binding assays is described in U.S. Pat. No. 3,720,760 which offers fluorescein derivatives, in particular fluorescein isothiocyanate, as a label candidate. Such an assay would employ conventional fluorometric techniques to monitor the label. The fluorescer would be excited by irradiation with light of appropriate wavelength and the fluoresced light, appearing at a different wavelength, measured. Various refinements and improvements of this basic fluorescent binding assay technique have evolved, as represented by the techniques described in U.S. Pat. Nos. 3,992,631; 3,999,948; 4,020,151; 4,025,310; 4,036,946; and 4,058,732. These fluorescent assays are based on a heterogeneous format, that is, the label is measured in the bound- or free-species after their separation.
Certain fluorescent binding assays of the homogeneous type have been conceived whereby the usually disadvantageous separation step can be avoided. One such method, the fluorescence polarization technique, is described in U.S. Pat. No. 4,115,699 and is based on the observation that irradiation with polarized light of certain fluorescer-ligand conjugates when bound by a binding partner (e.g., antibody) results in emission of light which has a different polarization. Thus, the bound and free-species of the labeled conjugate can be effectively distinguished in the monitoring reaction.
Another homogeneous fluorescent binding assay is based on quenching or enhancement of fluorescence upon binding of a fluorescer-ligand conjugate by its binding partner. Examples of these techniques are provided by the descriptions in Belgian Pat. No. 858,722 and German Offenlegungsschriften Nos. 2,716,276 and 2,716,515. A variation of this assay method is described in U.S. Pat. No. 3,996,345 which employs a specific quenching substance as a counterpart to the fluorescer label.
Chemiluminescent binding assays are the subject of U.S. Pat. No. 4,104,029 and U.S. patent applications Ser. Nos. 894,836 and 894,838, filed Apr. 10, 1978, which applications are assigned to the present assignee. As indicated previously, the chemiluminescent-based assays specifically described employ consumable reactants as the label, thereby theoretically limiting sensitivity, and, more significantly, are susceptible to protein quenching.
Of ancillary relevance to the present invention are the descriptions in U.S. Pat. No. 3,689,391; Rauhut et al, J. Am. Chem. Soc. 88:3604(1966); Rauhut et al, Accounts of Chem. Res. 2:80(1969); and Maulding et al, J. Org. Chem. 33:250(1968), relating to the inducement of photochemical reactions in the absence of external light by chemical, thermal or electrical excitation of a chemiluminescent material. Application of such phenomenon to analytical methods is not suggested in these references, which were published prior to the previously mentioned references relating to fluorescent immunoassays, or in the subsequent prior art.